The overall goal of our research is to understand the origin, development and regulation of the embryonic progenitor to the hematopoietic stem cell (HSC), hemogenic endothelium (HE). The focus of this proposal is to use human pluripotent stem cells (hPSCs) and mouse embryonic development as complementary model systems to identify the essential signals and genes for HE specification. Currently, the signal requirement(s) for HE specification from nascent mesoderm during embryonic development remains a poorly understood process. Our proposed studies build on our recent advances in the directed differentiation of definitive hematopoietic progenitors from hPSCs, having identified a WNT-dependent mesodermal population that harbors exclusively definitive hematopoietic potential, via CDX4 expression. We have now identified a novel subset of early mesoderm that is entirely dependent on retinoic acid (RA) signaling for the generation of HOXA+ HE with multi- lineage definitive hematopoietic potential. As RA is required for HE function during embryogenesis, we hypothesize that hPSC-derived RA-dependent HE is derived from a very unique mesodermal subset, which is ontogenically distinct from mesoderm harboring HSC-independent HE potential. We will test this hypothesis across 3 Specific Aims. In Aim 1, we will assess the physiological relevance of ALDH1A2+ derived HE, both in vitro and in vivo. The objective of these studies is to understand whether ALDH1A2+ mesoderm gives rise to HSC-competent HE. In Aim 2, we will define the mechanism the specification of RA-dependent HE. The objective of these studies is to determine how RA signaling affects HE development. In Aim 3, we will identify the role of alternative splicing during RA-dependent definitive hematopoietic specification. The objective of these studies is to define the essential transcriptional regulators within early mesoderm for the development of HE. The successful completion of these studies will provide us with a more comprehensive understanding of hematopoietic development. This is of fundamental importance to basic biology, and the insights generated from these studies will have clinical implications, such as the in vitro generation of HSCs for a wide array of regenerative medicine applications. Our unique cellular and molecular tools, combined with our expertise in hematopoietic development, stem cell biology and bioinformatics puts us in an ideal position to make a significant impact in this field.